Age-Specific Effects on Rat Lung Glutathione
and Antioxidant Enzymes after Inhaling Ultrafine Soot
Jackie K. W. Chan1, Sean D. Kodani1, Jessie G. Charrier2, Dexter Morin3, Patricia C. Edwards1,
Donald S. Anderson1, Cort Anastasio2, and Laura S. Van Winkle1,4
1
Center for Health and the Environment; 2Land Air and Water Resources; 3Department of Veterinary Medicine: Molecular Biosciences, and
Department of Veterinary Medicine: Anatomy, Physiology and Cell Biology, University of California at Davis, Davis, California
4
Vehicle exhaust is rich in polycyclic aromatic hydrocarbons (PAHs)
and is a dominant contributor to urban particulate pollution (PM).
Exposure to PM is linked to respiratory and cardiovascular morbidity
and mortality in susceptible populations, such as children. PM can
contribute to the development and exacerbation of asthma, and this
is thought to occur because of the presence of electrophiles in PM
or through electrophile generation via the metabolism of PAHs.
Glutathione (GSH), an abundant intracellular antioxidant, confers
cytoprotection through conjugation of electrophiles and reduction
of reactive oxygen species. GSH-dependent phase II detoxifying
enzymes glutathione peroxidase and glutathione S-transferase
facilitate metabolism and conjugation, respectively. Ambient particulates are highly variable in composition, which complicates systematic study. In response, we have developed a replicable ultrafine
premixed flame particle (PFP)-generating system for in vivo studies.
To determine particle effects in the developing lung, 7–day-old neonatal and adult rats inhaled 22 mg/m3 PFP during a single 6-hour
exposure. Pulmonary GSH and related phase II detoxifying gene
and protein expression were evaluated 2, 24, and 48 hours after
exposure. Neonates exhibited significant depletion of GSH despite
higher initial baseline levels of GSH. Furthermore, we observed attenuated induction of phase II enzymes (glutamate cysteine ligase,
glutathione reductase, glutathione S-transferase, and glutathione
peroxidase) in neonates compared with adult rats. We conclude that
developing neonates have a limited ability to deviate from their normal developmental pattern that precludes adequate adaptation to
environmental pollutants, which results in enhanced cytotoxicity
from inhaled PM.
Keywords: glutathione S-transferases; glutathione peroxidase; oxidative stress; lung development; ultrafine particulate matter
Urban ambient particulate aerosols are an aggregate of small
particles, liquid droplets, and vapors that has been linked to
respiratory and cardiovascular morbidity and mortality in susceptible populations (1, 2). Young children are especially susceptible to inhaled environmental pollutants. Factors that make
children susceptible include enhanced physical and aerobic
activities, larger body surface area to volume ratio, higher
(Received in original form March 19, 2012 and in final form September 18, 2012)
This work was supported by Cellular and Molecular Imaging Core Facility and the
inhalation exposure facility at the California National Primate Research Center
grant RR00169, by United States Environmental Protection Agency grant RD83241401–0, by STAR Fellowship Assistance Agreement no. FP-91718101–0
awarded by the USEPA (J.G.C.), by National Institute of Environmental Health
Sciences grant P42ES004699, and by a training program in Environmental Health
Sciences (grant T32 ES 007059) (J.K.W.C.).
Correspondence and requests for reprints should be addressed to Laura S. Van
Winkle, Ph.D., Department of Anatomy, Physiology and Cell Biology, School of
Veterinary Medicine., University of California-Davis, Davis, CA 95616-8732.
E-mail: lsvanwinkle@ucdavis.edu
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol Vol 48, Iss. 1, pp 114–124, Jan 2013
Copyright ª 2013 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2012-0108OC on October 11, 2012
Internet address: www.atsjournals.org
CLINICAL RELEVANCE
This study compares how glutathione and glutathionerelated enzymes are affected by combustion generated ultrafine particulate matter (PM) between neonatal and adult
rats in the lung. Knowing how this important antioxidant
pathway is affected upon PM exposure reveals important
age-specific differences in antioxidant enzyme expression,
which may help explain enhanced susceptibility to PM in
young children.
metabolic rate and minute ventilation, and increased oxygen
consumption compared with adult rats (3). Furthermore, the
lung continues to grow postnatally while undergoing alveolarization. This occurs on a backdrop of continuous differentiation
and maturation of critical cell types in the epithelium (4, 5) that
may be disrupted through environmental insults.
Vehicle exhaust from combustion of gasoline, diesel, and
other petroleum fuels is a dominant contributor to fine (PM2.5)
and ultrafine (PM0.1) particulates (6) and contains emissions of
carbonaceous particles with fused and free polycyclic aromatic
hydrocarbons (PAHs). PAH metabolism through phase I xenobiotic metabolism generates electrophilic and reactive metabolites that have been indicated as inducers of pulmonary cytochrome
P450 s in diesel exhaust particles (7, 8). Furthermore, ambient
PM contains persistent free radicals and reactive oxygen species (ROS) implicated in the generation of cellular oxidative
stress (9). Persistent oxidative stress contributes to decreased
lung function and increased diagnosis and exacerbation of
asthma, bronchitis, and pneumonia in children living in areas
of high levels of particulate air pollution (10, 11). Although
exposure to PM0.1 has been linked to diminished lung development and function in children (12, 13), the underlying biological
mechanisms responsible for enhanced susceptibility remain
elusive.
Although many nonenzymatic antioxidants are present in the
lung, glutathione (GSH) is the most abundant antioxidant, produced in millimolar concentrations in the cell. It is essential for
cellular protection through the conjugation of electrophiles and
reduction of ROS. GSH is a cofactor for the selenoenzyme glutathione peroxidase (GPX1) and is a cosubstrate in reactions
facilitated by glutathione S-transferases (GST). GSH can be
replenished through reduction of glutathione disulfide (GSSG)
by glutathione reductase (GSR) or through biosynthesis via
the g-glutamyl cycle (14). The g-glutamyl cycle consists of two
ATP-dependent steps, where amino acids L-cysteine, L-glutamic
acid, and glycine are combined to form the GSH tripeptide. The
first, rate-limiting step combines L-glutamate and cysteine to
form g-glutamylcysteine via the enzyme glutamate cysteine ligase (GCL) before adding glycine to generate GSH. GCL is an
oxidant-sensitive heterodimer composed of a catalytic (GCLC)
Chan, Kodani, Charrier, et al.: Age Determines Glutathione Response to PM
and a regulatory (GCLM) subunit. Both have been shown to be
up-regulated after oxidative stress (15, 16).
Field studies indicate the composition of ambient PM is
highly variable and is dependent on the time, day, weather,
and location. The variable nature of ambient PM complicates
the systematic study of health effects, so we have developed
and characterized a premixed flame particle (PFP)-generating
system (17, 18) for in vivo chamber inhalation exposure studies.
Ethylene flame–generated PFPs are 70-nm ultrafine particles
rich in PAHs that are also present in the vapor phase. It has
previously been shown that, although a quarter of deposited
ultrafine particles are cleared by mucoiliary clearance within
24 hours after exposure, a significant fraction of particles are
retained within the lungs even after 48 hours (19). In the current
study, we exposed male, 7-day-old neonatal pups and 8-week-old
young adult rats to a single acute inhalation exposure to PFPs
and collected samples at various times up to 48 hours after exposure. We have previously shown that neonates have enhanced
susceptibility to PFPs compared with adult rats (18). To further
investigate possible mechanisms responsible for the enhanced
cytotoxicity, we analyzed GSH levels as well as biosynthesis and
conjugating enzymes related to the glutathione pathway (Figure 1).
We hypothesized that basal differences and responses between
neonates and adult rats would play a role in the enhanced neonatal susceptibility to PFP. The objectives of this study were (1)
to quantify basal GSH and GSSG levels between adult rats and
neonates and their responses after exposure, (2) to define the
pattern of expression of key GSH resynthesis enzymes after PM
oxidative stress, and (3) to describe differential responses of conjugating enzymes GPX1 and GSTs.
MATERIALS AND METHODS
Flame and Particle Generation
115
microdissection and prepared for HPLC analysis of GSH and GSSG as
described (20), with alterations listed in the online supplement.
RNA Isolation and Real-Time PCR
Microdissected lung compartmental RNA was isolated (21). Gene
expression was determined using Taqman probes and primers (Applied
Biosystems, Foster City, CA) (21, 22) listed in Table 1. Results
were calculated using the comparative-Ct method (23, 24) with
hypoxanthine-guanine phosphoribosyltransferase as the reference gene
(18, 25). Results are expressed as fold changes relative to filtered animals of the same age unless otherwise stated.
Immunohistochemistry
Lungs were inflated with 37% formaldehyde vapor bubbled under 30 cm
hydrostatic pressure for 1 hour and embedded in paraffin within 24 hours
(26, 27). Paraffin sections from all groups were stained simultaneously
to minimize variability and were immunostained for GSR (Abcam,
Cambridge, MA) at 1:500, GPX1 (Abcam) at 1:2,000, and GCL (Neomarkers, Fremont, CA) at 1:300. The concentration of primary antibody
was determined through a series of dilutions to optimize for staining
density while minimizing background (25, 28).
Western Blotting
Flash-frozen lung tissue was homogenized in RIPA lysis buffer (Santa
Cruz Biotechnology, Santa Cruz, CA), and protein concentrations were
determined with the Bradford assay (Bio-Rad, Hercules, CA) using
BSA as standards. Samples were reduced for SDS-PAGE, and 20 to
40 mg protein per lane was electrophoresed and probed against
GPX1, GSR, GCL, and Actin (details are provided in the online
supplement). Bands were quantified using ImageJ software (NIH,
Bethesda, MD).
Hydrogen Peroxide Assay
PFPs were burner generated (17, 18) at a mass concentration of 22.4 6
5.6 mg/m3 PFP (mean 6 SD) with a mean particle size of 70.6 nm 6 1.5.
Particles were high in organic carbon with an lelemental carbon:organic
carbon ratio of 0.58.
Hydrogen peroxide (H2O2) production from PFP was measured in
triplicate in an in vitro, cell-free surrogate lung fluid containing ascorbate, citrate, glutathione, and urate (29). H2O2 was quantified using
HPLC with fluorescence detection (30). Details are provided in the
online supplement.
Animal Exposure Protocol
Statistics
Eight-weekold male adult rats and newborn postnatal male Sprague
Dawley rats with dams (Harlan Laboratories, Indianapolis, IN) were
acclimated in filtered air (FA) for 5 to 7 days before use as previously
described (17, 18). Animals were necropsied at 2, 24, and 48 hours after
cessation of the 6-hour exposure to FA or PFP.
All data are reported as mean 6 SEM unless otherwise stated. Statistical outliers were eliminated using the extreme studentized deviate
method. Undetected and samples below detection limit were imputed
using natural-log regression on order statistics (lnROS) (31, 32). Multivariate ANOVA (MANOVA) was applied against age, compartment,
and exposure factors when appropriate. Pairwise comparisons were
performed individually using one-way ANOVA followed by Fisher’s
protected least significant difference post hoc analysis using StatView
(SAS, Cary, NC). P values of , 0.05 were considered statistically
significant.
HPLC
Lungs were inflated with a solution of 1% agarose (Sigma Chemical, St.
Louis, MO). Airways and surrounding parenchyma were separated by
Figure 1. Diagram of glutathione cycle and proposed premixed flame particle (PFP) metabolism indicating endpoints analyzed in this study. PFP directly or indirectly,
through the generation of electrophiles, produces hydrogen peroxide and other reactive oxygen species. Glutathione (GSH) conjugates these reactive species directly or
through the glutathione S-transferase (GST) enzymes.
GSH can be generated through two separate pathways:
de novo through the g-glutamyl cycle with glutamate cysteine ligase (GCL) as the rate-limiting enzyme or via reduction of glutathione disulfide through GSR. Bolded portions
of these pathways were analyzed in the current study.
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TABLE 1. TAQMAN GENE EXPRESSION ASSAY CATALOG NUMBERS
Symbol
Assay ID
Gene Name
NCBI RefSeq
GCLc
GCLm
GPX1
GSR
GSTM1
GSTP1
GSTT1
HPRT
Rn00563101_m1
Rn00568900_m1
Rn00577994_g1
Rn01482159_m1
Rn00755117_m1
Rn00561378_gH
Rn00583932_m1
Rn01527840_m1
Glutamate-cysteine ligase catalytic subunit
Glutamate-cysteine ligase regulatory subunit
Glutathione peroxidase
Glutathione reductase
Glutathione S-transferase Mu-1
Glutathione S-transferase Pi-1
Glutathione S-transferase Theta-1
Hypoxanthine-guanine phosphoribosyltransferase
NM_012815.2
NM_017305.2
NM_030826.3
NM_053906.2
NM_017014.1
NM_012577.2
NM_053293.2
NM_012583.2
RESULTS
GSH and GSSG
We measured levels of the ubiquitous antioxidant GSH and its
oxidized dimerized form (Figure 2). We used MANOVA to
analyze whether age (neonates versus adult rats), compartment
(airway versus parenchyma), or exposure (FA versus PFP2 or
PFP24) were contributing factors to measured differences in
GSH levels. We collected time-matched FA and PFP samples
at 2 and 24 hours after exposure. GSH levels were similar across
FA2 and FA24 in neonates and adult rats and were pooled for
analysis. GSH levels differed significantly by age (P ¼ 0.0002),
with 4- to 5-fold higher GSH concentrations in neonates. The
MANOVA value for the compartment factor (P ¼ 0.0074) signified elevated parenchymal GSH. Neonates had approximately
5 times more GSH in airways and parenchyma compared with
adult rats. GSH levels were similar between compartments
within each age. After PFP exposure, an abrupt drop in GSH
was observed in PFP24 neonates in airway (P ¼ 0.017) and
parenchymal (P ¼ 0.0074) compartments compared with FA
controls (Figure 2A). Adult rats showed an opposite response;
we observed a time-dependent increase of GSH concentration
in the parenchyma that reached significance in PFP24 (P ¼
0.036). Airway GSH levels remained unchanged after exposure
(Figure 2B). Multivariate analysis on GSSG concentrations did
not convey any significant main effects. Pairwise comparisons
revealed significantly higher basal GSSG levels in neonate airways compared with adult rats. A precipitous drop in airway
GSSG was observed in neonates after PFP exposure in the
PFP24 group, compared with FA controls (P ¼ 0.032) and
PFP2 (P ¼ 0.0058). Neonatal parenchymal GSSG remained
unchanged after exposure (Figure 2C). Adult parenchymal
GSSG showed a time-dependent increase in response to PFP
exposure. Parenchymal GSSG in the PFP24 group was elevated
significantly compared with the FA control (P , 0.0001) and
PFP2 (P , 0.0001) groups. Contrary to neonates, airway GSSG
was unaffected by PFP exposure (Figure 2D).
Glutamate Cysteine Ligase
To investigate particle effects on GSH regeneration, we analyzed
expression of glutamate GCL, the rate-limiting enzyme in GSH
biosynthesis (14). We measured mRNA expression levels for
the catalytic (GCLC) and regulatory (GCLM) subunits using
RT-PCR in the airway and parenchymal lung compartments in
7-day postnatal and adult animals (Figure 3). We used MANOVA
to determine whether age, compartment, and exposure (FA
versus PFP2, PFP24, or PFP48) factors played a role in GCLC
and GCLM expression. We found multiple interactions, with
a significant effect of age (P , 0.0001); GCL expression was
greatest in adults. Furthermore, there was a main effect in compartment (P , 0.0001), signifying greater airway expression.
A complete table of pairwise comparisons in FA controls
showing P values and significant up- or down-regulation is
available in the online supplement (see up and down arrows
in Table E1 in the online supplement). Although GCLM expression remained relatively constant across age and compartments, GCLC expression was 2-fold greater in the airways
compared with parenchyma in both ages. Adults generally had
a 1.7-fold greater GCLC expression than neonates, with the highest levels in the airways (Figure 3A). In neonates, we observed
a transient drop in airway GCLC expression at PFP24 compared
with PFP2, whereas parenchymal and GCLM expression
remained unchanged after PFP exposure (Figure 3B). In comparison, there were no significant exposure effects for GCLC or
GCLM expression in each lung compartment in adult animals
(Figure 3C).
GCLC and GCLM protein expression was quantified using
Western blotting (Figure 4A). Pairwise comparisons revealed
no exposure effects in either subunit in neonates (Figure 4B),
but a transient up-regulation in GCLC expression was observed
in the adult PFP2 group (P ¼ 0.048) compared against FA
controls. Adult GCLM expression was also unaffected by PFP
exposure (Figure 4C). We performed immunohistochemistry to
determine spatial localization of the protein in the lung tissue.
We observed intense GCL staining in airway epithelium and
parenchyma in PFP2 groups of neonates (Figure 4E) and adult
rats (Figure 4I) compared with FA controls. Although neonatal
GCL expression reverted to FA distribution and abundance at
PFP24 and PFP48 (Figures 4F and 4G), adult GCL expression
remained elevated and was localized in the airway epithelium
(Figures 4J and 4K).
GSR
Next, we examined the other arm of GSH regeneration:
NADPH-mediated reduction of GSSR back to GSH via GSR
(Figure 5). Gene expression and MANOVA revealed little difference in GSR expression against age, exposure, and compartment factors. We discovered a single difference: adult airways
had significantly greater GSR expression after PFP48 compared
with FA controls (P ¼ 0.0077). GSR protein expression (Figure
5D) was relatively similar to the RT-PCR results. Although
GSR protein abundance was unchanged after PFP exposure
(Figure 5E), we saw an almost significant increase in the adult
PFP48 group compared against FA controls (P ¼ 0.06) (Figure
5F). Immunohistochemical localization of GSR showed mirrored trends from the Western blots. We did not see any
GSR protein in FA neonates (Figure 5G). After exposure, the
observed transient GSR staining in the bronchiolar epithelium
in PFP2 (Figure 5H) returned to FA steady state by PFP24 and
PFP48 (Figures 5I and 5J). Adult animals have more abundant
basal GSR staining compared with neonates (Figure 5K), which
was also enhanced after PFP exposure. Starting at PFP2, we
detected robust bronchiolar epithelial staining, which persisted
to PFP48 (Figures 5L–5N). Furthermore, several intensely
stained cells within the parenchymal tissue could be seen solely
in the PFP48 group.
Chan, Kodani, Charrier, et al.: Age Determines Glutathione Response to PM
117
Figure 2. GSH (A and B) and
glutathione disulfide (GSSG)
(C and D) concentrations were
measured in microdissected
airway (white bars) and parenchymal (gray bars) tissue compartments using HPLC with
electrochemical detection in
neonatal (A and C) and adult
(B and D) rats. Overall, neonates exposed to filtered air
(FA) had approximately 5-fold
greater GSH in both lung compartments compared with
adult rats. After PFP exposure,
neonatal GSH levels were significantly depressed in PFP24
compared with FA and PFP2
(A). Adult GSH, however,
showed an increase in parenchymal GSH in PFP24 (B).
GSSG levels followed a similar
trend. Neonatale airway GSSG
was markedly dropped in
PFP24 (C), whereas adult parenchymal GSSG experienced
a time-dependent increase
(D). Data are plotted as
means 6 SEM (n ¼ 6–12 rats
per group, per compartment). P , 0.05 are denoted
as follows: *significantly different from neonates in the same compartment, ysignificantly different from airways in the same exposure and
age, and zsignificantly different from FA in the same compartment and age.
GPX1
GPX1 decreases reactive hydroperoxides using glutathione as a
cosubstrate (33), making it a suitable candidate to analyze the
effects of ROS on glutathione-dependent antioxidant enzymes
(Figure 6). Using RT-PCR and performing MANOVA against
age, exposure, and compartment factors, we detected significant
age (P , 0.0001), exposure (P ¼ 0.0031), and compartment (P
¼ 0.0007) effects. Adults had a significantly higher GPX1 expression, which was increased by PFP exposure and was greater
in parenchyma compared with in airways. To determine the
specific differences, we applied pairwise comparisons against
individual groups. Although GPX1 expression was similar between the airway and parenchyma compartments in neonates,
adult parenchymal GPX1 expression was significantly greater
than adult airways (P ¼ 0.035) and neonatal parenchyma
(P ¼ 0.016) (Figure 6A). After PFP exposure, neonatal GPX1
expression was significantly down-regulated in the PFP48 group
in airway and parenchyma compartments compared with the
PFP24 group (Figure 6B). A divergent trend was observed in
adult rats. Compared with FA controls, we detected significant
GPX1 up-regulation in the parenchyma in PFP24 as well as in
airways and parenchyma in the PFP48 groups (Figure 6C).
Because GPX1 is responsible for reducing hydroperoxides,
we measured the rate of H2O2 production in vitro in a cellfree surrogate lung fluid. We observed substantially more
H2O2 in the filters containing PFP compared with filter blank
controls (Figure 6D). Additionally, we found that PFP particles generate a similar amount of H2O2 compared with ambient particulates collected from Fresno, California on a massnormalized basis (see Figure E1 in the online supplement).
Next, we analyzed GPX1 protein expression (Figure 6E) to
correlate with our previous results. Generally, GPX1 protein
is consistent with the mRNA expression data. Although GPX1
abundance was largely unchanged in the neonates (Figure 6F),
we found a significant time-dependent increase in adults, where
PFP24 (P ¼ 0.045) and PFP48 (P ¼ 0.023) groups had significantly elevated GPX1 levels compared with FA controls (Figure
6G). Immunohistochemistry localized GPX1 protein to airway
epithelium and parenchyma of neonates and adult rats in FA
controls, but neonates exhibited a lower overall abundance (Figures
6H and 6L). After PFP exposure, GPX1 remained unchanged
in neonates (Figures 6I–6K). In adult rats, PFP induced GPX1
throughout the lung tissue. Staining was darker in all lung compartments; intensely positive staining was observed in bronchiolar
epithelium (Figures 6M–6O, arrows) regardless of the time after
exposure.
GST
We evaluated mRNA expression of the m (GSTM1), p (GSTP1),
and u (GSTT1) isoforms in the GST family (Figure 7), which
catalyze the conjugation of glutathione to electrophilic substrates as a contributor to phase II xenobiotic biotransformation (34). We applied MANOVA against age, compartment,
and gene (GSTM1 versus GSTP1 versus GSTT1) factors on FA
controls and found significant effects (P , 0.0001) in all three
main factors (age, compartment, and gene). Pairwise comparisons among GSTs in FA controls are presented in Table E2.
Under basal conditions, GSTM1 is expressed 4- to 5-fold higher
in airways compared with parenchyma; these findings were consistent between adult rats (P , 0.0001) and neonates (P ¼
0.006). Similarly, GSTM1 is 3- to 4-fold higher in adults across
compartments, with GSTM1 maximally expressed in adult airways. GSTP1 was the most abundant GST among the three
isoforms comparing between ages with the exception in adult
airways, where GSTM1 was most abundant. Adult rats had
significantly greater GSTP1 expression in airway (P ¼ 0.021)
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 48 2013
was expressed at levels 2-fold higher in adults than neonates and
in the airways than in the parenchyma (Figure 7A). After PFP
exposure, GSTM1 expression was significantly elevated in PFP48
adult airways compared with all other exposure time points. Besides a transient increase in neonatal airway PFP2 compared with
PFP48, no other age or compartment had significant exposurerelated effects (Figure 7B). GSTP1 expression revealed a similar
pattern; adult airway expression in PFP48 was significantly greater
than all other exposures. However, a transient decrease in GSTP
was observed in adult parenchyma in the PFP2 group (P ¼ 0.049)
that recovered at later time points. Neonatal expression did
not differ from FA controls after PFP exposure, but the PFP24
and PFP48 groups were depressed compared against PFP2 in
the parenchyma (Figure 7C). Finally, GSTT1 expression was
down-regulated in adult PFP24 parenchyma (P ¼ 0.029) compared with adult FA controls. No other age or compartment
had significantly changed from their respective FA controls
(Figure 7D).
DISCUSSION
Figure 3. GCLc and GCLm gene expression. RT-PCR expression in airway and parenchyma compartments in neonates and adult rats exposed to PFPs. Basal GCLc was consistently expressed in higher
abundance than GCLm, and its highest expression was seen in adult
airways (A). After PFP exposure, a transient drop in neonatal airway
GCLc expression was observed in PFP24 compared with PFP2 (B). No
treatment effects were detected in adult animals (C). Data are plotted
as means 6 SEM (n ¼ 5–7 rats per group, per compartment, per gene).
P , 0.05 are denoted as follows: *significantly different from neonates
in the same compartment, and ysignificantly different from airways in
the same age. PFP2, PFP24, and PFP48 refer to PFP exposure for 4, 24,
and 48 hours, respectively.
and parenchyma (P ¼ 0.016) compared with neonates, but there
were no differences observed between compartments in both
age groups. GSTT1 was the least abundant GST isoform but
In the current study, we used site-specific approaches to define
GSH and GSSG concentrations as well as expression patterns for
a number of antioxidant gene and proteins to identify the underlying cause of enhanced susceptibility in neonatal versus adult
rats. To determine how conducting airways and alveoli separately respond to PFP exposure, airways were microdissected
from alveolar parenchyma to evaluate compartment-specific
responses. We have previously reported that neonates are more
susceptible to inhaled PFPs than adults based on elevated LDH
leakage and ethidium homodimer-1 staining (18), which has
correlated well with the literature on neonatal susceptibility to
particulate matter (35, 36). Moreover, we have shown that subchronic exposure to PFPs during this period of development
causes decrements in airway growth that are not recovered
upon reaching adulthood (17). In other exposure models, inhalation of ultrafine particulates increases airway resistance and
decreases compliance (37), suggesting impaired lung function.
We have built upon our previous work by analyzing components of the phase II detoxification pathway, namely GSHrelated protective responses. We measured levels of the ubiquitous antioxidant GSH, the GSH biosynthetic enzyme GCL, the
GSH regeneration enzyme GSR, the GSH selenoenzyme GPX1,
and several isoforms of GST conjugation enzymes. In a majority
of these measures, we have noted that neonates remained essentially unchanged after exposure, in striking contrast to an overall
up-regulation of the aforementioned antioxidants observed in
adults after inhalation of particles. We demonstrated a unique
failure of antioxidant proteins in 7-day postnatal rats to respond
to an acute, single 6-hour inhalation exposure to an atmosphere
containing flame-generated ultrafine particles. This response differed markedly from the effective up-regulation of these antioxidant responses in adult rats.
PFP is an ethylene flame–generated ultrafine carbonaceous
soot. It is rich with attached and free PAHs and closely resembles diesel exhaust with a low elemental carbon:organic carbon
ratio of 0.58. Methylated biphenyls and substituted naphthalenes are the dominant organic species measured, but larger
PAHs, such as fluorene, phenanthrene, pyrene, and benzopyrenes, were also detected in subnanogram quantities (18). Our
data revealed that these particles are potent hydrogen peroxide
producers and are similar in their peroxide-generating capability to ambient particles collected in Fresno, California. Furthermore, the PFP chamber particle concentration ([9.4 6 0.5] 3
10 4 particles/cm 3 ) was similar to values reported 30 m
downwind from a major highway in Los Angeles, California
Chan, Kodani, Charrier, et al.: Age Determines Glutathione Response to PM
119
Figure 4. GCLc and GCLm protein analysis through Western blotting and immunohistochemistry. Representative GCLc and GCLm Western blots
with actin loading control (A). GCLc/m Western blots were quantified: while neonatal GCLc/m expression remained unchanged after exposure (B),
a significant up-regulation in GCLc was detected in PFP2 adult rats compared against FA controls (C). Data are plotted as means 6 SEM (n ¼ 6 rats
per group). P , 0.05 is denoted as follows: zsignificantly different from FA in the same age. GCL immunohistochemical images in neonatal (D–G)
and adult (H–K) rats reared in FA (D and H) and exposed to PFP: PFP2 (E and I), PFP24 (F and J), and PFP48 (G and K). Intense GCL staining was
observed in adult and neonatale PFP2. In contrast to neonates, staining in adult PFP48 was continually up-regulated. High magnification inserts
highlight GCL-positive cells in treated groups. Scale bars for D–K (shown in K) are 50 mm.
(z 5.0 3 104 particles/cm3; 65-nm particles) (38). Our chamber
dose (22.4 mg/m3) is well below the 2006 United States Environmental Protection Agency revised 24-hour average National Ambient Air Quality Standards for PM2.5 of 35 mg/m3, highlighting
the importance of using supplementary measurements in addition
to PM mass to determine ultrafine particulate contributions to
aerosol mass. Our current study shows that neonates respond
very differently than adult rats to environmentally relevant
levels of fine PM.
Although many antioxidants are present in the rat lung, including a variety of vitamins and ascorbic and uric acids, we limited our analysis to the most ubiquitous antioxidant GSH. We
found that neonatal GSH levels were several-fold greater than
in adult rats regardless of compartment. This is expected due
to initial adaptation to a hyperoxic environment at birth. GSH
depletion via buthionine sulfoximine in newborn rats has been
shown to be lethal due to the accumulation of endogenous
ROS generated from mitochondrial metabolism that induces oxidative stress and injury (39). Although GSH and GSSG results
are highly variable among published studies using a variety of
methods, our data fall within the ranges (0.54–15.2 nmol/mg
protein GSH and 0.069–7.66 nmol/mg protein GSSG) reported
previously in control rat lung homogenates (40–42). Our findings are in accordance with previously published data using lung
microdissection approaches coupled with HPLC electrochemical detection and fluorescence methods in the rat lung that
yielded 4.4 to 9.1 nmol/mg protein airway GSH and 10.26 to
10.6 nmol/mg protein parenchymal GSH (43, 44). After PFP
exposure, we observed disparate trends between the two age
groups. Adult GSH and GSSG concentrations were elevated
24 hours after exposure solely in the parenchymal compartment. Our findings are in accordance with data reported by AlHumadi and colleagues, who showed elevated levels of GSH in
alveolar macrophages from adult Sprague Dawley rats intratracheally instilled with diesel exhaust particles (45). However,
neonatal levels of GSH and GSSG were depressed in the airways. Differences in GSH and GSSG levels between lung compartments and their differential responses to PFP exposure
highlights the heterogeneity of the lung, where specific cell populations exist in distinct microenvironments. GSH depletion has
been linked with epithelial cytotoxicity, where decrements below a certain threshold cause irreversible injury (46, 47). This
correlates well with the compartment-specific cytotoxicity we
found in our previous study (18). Additionally, the GSH:GSSG
ratio, typically considered an indicator of oxidative stress, was
significantly diminished in neonates after exposure but remained unchanged in adult rats. The declining trends in GSH
and GSSG after PFP exposure in neonates suggests a failure
to up-regulate or regenerate glutathione pools in very young
animals.
We analyzed mRNA and protein expression of several
glutathione-dependent enzymes: GCL, GSR, GPX1, and several
isoforms of GST. These antioxidant enzymes are regulated by
the nuclear factor erythroid-derived inducer 2 transcription factor under the antioxidant response element and are activated after exposure to oxidant stressors such as particulate matter (48).
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 48 2013
Figure 5. GSR gene and protein expression. Basal GSR gene expression was similar between age and compartments (A). No exposure-related gene
expression differences were observed in neonates after PFP exposure (B). In adult rats exposed to PFPs, a time-dependent increase of GSR gene
expression was observed 48 hours after exposure (C). Data are plotted as means 6 SEM (n ¼ 6 rats per group, per compartment). P , 0.05 is
denoted as follows: zsignificantly different from FA in the same compartment and age. A representative blot of GSR protein with actin as a loading
control (D). Quantification of GSR Western blots revealed no exposure-related effects in neonates (E). In adult animals, there was a strong but
insignificant trend 48 hours after PFP exposure (F). Protein data are mean 6 SEM (n ¼ 5 or 6 rats per group). Spatial immunohistochemical
localization of GSR in neonatal (G–J) and adult (K–N) of FA controls (G and K), PFP2 (H and L), PFP24 (I and M), and PFP48 (J and N) groups. A
transient up-regulation was seen in neonatal airways in PFP2, whereas adult animals had continual induction of GSR until PFP48. At PFP48, densely
stained cells were observed in the parenchyma (denoted by arrows). Scale bar for G–N (shown in G) is 50 mm.
To quantify the potential for GSH regeneration, we evaluated
the GCLC and GCLM subunits along with GSR mRNA transcript and protein expression levels. Although basal compartmental and age differences were apparent between the catalytic
and regulatory subunits, there were no exposure effects in
GCLC/M expression. Our data contrast with published reports
demonstrating GCLC/M mRNA up-regulation after ultrafine
particulate exposure (49–51). A possible explanation for the
disparity may be due to different exposure conditions. Our exposure concentrations were substantially lower (22.4 mg/m3 for
a single 6-h PFP exposure), compared with these past studies,
which used higher doses and longer exposure durations (4-d exposure to 5.0 mg/m3 ultrafine butadiene-generated soot [49],
8-wk exposure to 100 mg/m3 diesel exhaust particulates [50], or
10 wk of reaerosolized ultrafine ambient particulates [51] in
mice). Although we did not detect exposure effects in GCLC/M
mRNA expression, GCLC protein was elevated after exposure,
and this finding is similar to results recently published by Zhang
and colleagues, who presented similar GCLC up-regulation
results in 3-month-old “young adult” mice after chronic ambient
ultrafine particulate (,200 nm) exposure (51). A similar trend
is seen with GSR mRNA and protein expression. Although we
Chan, Kodani, Charrier, et al.: Age Determines Glutathione Response to PM
121
Figure 6. GPX1 gene and protein expression. GPX1 gene expression was greatest in the adult parenchyma; all other age and compartments had
similar expression levels (A). A significant reduction in GPX1 expression was observed in the PFP48 neonates compared with PFP24 in airways and
parenchyma (B). Contrary to neonates, GPX1 was up-regulated in adult rats. Parenchymal expression was significantly higher in the PFP24 and
PFP48 groups compared with FA controls and with the PFP2 group. Airway expression was significantly higher only in the PFP48 group (C). Data are
plotted as means 6 SEM (n ¼ 6 rats per group, per compartment). P , 0.05 is denoted as follows: *significantly different from neonates in the same
compartment, ysignificantly different from airways in the same age, and zsignificantly different from FA in the same compartment and age. The rate
of H2O2 production from PFP was significantly greater than from filter blanks (D). Data are plotted as means 6 SD (n ¼ 3). P , 0.05 is denoted as
follows: *significantly different from filter blank. GPX1 protein was quantified using Western blotting; a representative blot with actin-loading control
is shown (E). Quantification of GPX1 Western blots revealed no exposure-related effects in neonates (F) nut revealed a time-dependent increase in
GPX1 expression in adults after PFP exposure (G). Protein data are presented as mean 6 SEM (n ¼ 5 or 6 rats per group). P , 0.05 is denoted as
follows: zsignificantly different compared with FA controls. Spatial expression of GPX1 in neonatal (H–K) and adult rats (L–O) and of FA controls (H and L)
and PFP2- (I and M), PFP24- (J and N), and PFP48- (K and O) exposed groups. GPX1 induction in adults was seen in airway epithelium and parenchyma.
Arrows identify intensely stained bronchiolar epithelium present at all time points after PFP exposure. Scale bar for H–O (shown in N) is 50 mm.
did not observe any exposure-related mRNA or protein changes
in the neonates, we saw significant increases in adult airway
mRNA expression and a strong trend in protein expression in
the PFP48 group. These data are similar to previously published data showing enhanced GSR activity in alveolar macrophages of rats intratracheally instilled with diesel exhaust particles
(45). The inability of neonates to up-regulate this rate-limiting
enzyme, in addition to GSH depletion, may lead to the loss of
cellular homeostasis, where ROS generation overwhelms antioxidant defenses, resulting in oxidative stress. This may partially
explain why the neonatal rats are more susceptible to an acute
PM exposure compared with adult rats.
Cellular GPX1 is a phase II enzyme that confers cytoprotection through metabolism of H2O2 and organic peroxides (present endogenously and produced from deposited particulates)
that cause protein and lipid peroxidation, generating oxidative
stress. Superoxide anion, present in diesel exhaust particulate
extracts (52), is a substrate for superoxide dismutase, which
converts superoxide into H2O2 for GPX1 metabolism. GPX1
is a potent, selenium-dependent antioxidant that consumes GSH
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 48 2013
=
Figure 7. Expression of glutathione S-transferase isoforms (GSTM ¼
SGT m isoform; GSTP ¼ GST p isoform; and GSTT ¼ GST t isoform).
GSTM had widely variable compartmental and age expression, whereas
GSTP had age-specific differences. GSTT was the least abundant isoform but is expressed higher in adults airways (A). GSTM expression
was significantly up-regulated in adult airways in the PFP48 group
compared with all other exposure time points (B). GSTP expression
was similar: adult airways in the PFP48 group were also significantly
elevated against FA controls and exposure groups. A transient drop in
adult parenchyma was observed in PFP2 compared with FA controls
(C). GSTT expression was temporarily down-regulated in adult parenchyma in PFP24 (D). Data are plotted as means 6 SEM (n ¼ 5–7 rats
per group, per compartment, per gene). P , 0.05 are denoted as
follows: *significantly different from neonates in the same compartment, ysignificantly different from airways in the same age and
gene, and zsignificantly different from FA in the same compartment
and age. To reduce confusion among comparisons between groups,
superscript AP denotes comparison against adult parenchyma.
to reduce these oxidants to preserve cellular homeostasis (33, 53).
In the current study, we showed that PFP particles actively generate H2O2 in a cell-free surrogate lung fluid and observed timedependent elevation of GPX1 mRNA and protein expression
after exposure in adult animals. Our results are in agreement
with previous work that demonstrated GPX1 elevation in adult
rats 24 hours after a week-long exposure to cigarette smoke (54).
However, in comparison to adult animals, neonates did not reveal
the same pattern, and gene transcription and protein expression
remained unchanged after inhaling PFPs. The inability to respond
could increase neonatal vulnerability to PFP by increasing the
burden of intracellular H2O2, especially because GPX1 activity
is lowest at 6 to 16 days of age (55).
GSTs are a family of phase II detoxification enzymes that catalyze GSH conjugation with electrophilic compounds (34). We
focused on the m (GSTM1), p (GSTP1), and u (GSTT1) isoforms. These isoforms have significance in antioxidant defense;
previous clinical studies have shown adverse allergic responses
after inhalation of environmental pollutants in polymorphic or
null cohorts (56, 57). GSTM1 and GSTP1 expression was significantly up-regulated only in adult airways at 48 hours after
PFP exposure, again highlighting the importance of evaluating
compartment-specific effects. Our results are in accordance with
previously published data showing specific GSTM1 and GSTP1
induction in airways after treatment with nuclear factor
erythroid-derived inducer 2 (3)-tert-butyl-4-hydroxyanisole (58).
The failure of neonates to mirror adult responses is concerning
because it indicates an inability to up-regulate these protective
molecules in the face of a relevant environmental provocation.
Children with the GSTM1 null allele or GSTT1 deficiency have
increased risk of developing asthma and exacerbation of asthma
symptoms, such as wheezing or shortness of breath, after environmental tobacco smoke exposure (59). Furthermore, GSTP1 is
critical in the deactivation of cytochrome P450 metabolically activated PAHs present in PFPs. GSTP1 knockout mice are shown
to have increased DNA adducts and decreased GSH conjugates
after PAH treatment (60). Although GST proteins are detectable
by immunochemical methods at 7 days of age, GST–mediated
1-chloro-2, 4-dinitrobenzene conjugation activity is seen only 20%
of adults (61). This further highlights the importance of GSTs in
the defense against oxidant stressors in the developing lung. The
inability to adequately up-regulate key antioxidant enzymes
reduces the ability to detoxify electrophilic compounds present
on or in ultrafine particles, which may enhance cytotoxicity and
increase susceptibility in neonates, who are already at a disadvantage due to the postnatal maturation of this enzymatic activity.
Chan, Kodani, Charrier, et al.: Age Determines Glutathione Response to PM
This study shows that a short-term, low-dose inhalation exposure to an environmentally relevant level of combustion-derived
ultrafine particles causes up-regulation of glutathione and
glutathione-related enzymes in the adult rat. We hypothesize
that failure to up-regulate key enzymes is due to a limited availability to deviate from a normal developmental pattern of expression in the postnatal animal. Our data strongly support
the notion that the appropriate age group, in our case 7-dayold neonates, be used when evaluating the potential effect of
an environmental pollutant on susceptible populations such
as young children. We have shown that neonates suffered a precipitous drop in GSH concentrations after PFP inhalation that
was not regenerated by GCL or GSR. Furthermore, phase II
enzymes such as GSTs and GPX1 remained unchanged after exposure, possibly as a result of the lack of GSH as a reducing substrate, which potentially enhances cytotoxicity in neonates. We
conclude that, compared with mature adult rats, the developing
lung fails at up-regulating key GSH regeneration and antioxidant
enzymes to adequately adapt to ultrafine particulate exposure.
The downstream effects may result in enhanced cellular injury
and oxidative stress compared with adult rats exposed to the
same dose.
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgments: The authors thank Brian Tarkington, Ashley Cooper, Louise
Olson, Judy Shimizu, Aamir Abid, and Christopher Wallis for their skilled technical
assistance during exposures, sample collection, and processing; Jonathan Rutherford for his expertise in drawing vector graphics; and Dr. Alan Buckpitt for reading
and editing this manuscript.
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